The major carbohydrates found in vascular plants are sucrose, starch, cellulose and fructans. Sucrose is most commonly purified from sucrose-producing plants and used as a sweetener. Starch and cellulose are currently used in numerous food and non-food applications in their native form or after chemical modification or hydrolysis. Fructans have commercial applications in the industrial, medical, food and feed industries.
Starch is a mixture of two polysaccharides, amylose and amylopectin. Amylose is an unbranched chain of up to several thousand α-D-glucopyranose units linked by α-1,4 glycosidic bonds. Amylopectin is a highly branched molecule of up to 50,000 α-D-glucopyranose residues linked by α-1,4 and α-1,6 glycosidic bonds. Approximately 5% of the glycosidic linkages in amylopectin are α-1,6 bonds, which leads to the branched structure of the polymer.
Amylose and amylopectin molecules are organized into granules that are stored in photosynthetic tissues during light periods or in storage organs. The ratio of amylose to amylopectin and the degree of branching of amylopectin affect the physical and functional properties of the starch. Functional properties, such as viscosity and stability of a gelatinized starch, determine the usefulness and value of starches in food and industrial applications. Currently, specific functional properties are met by using starches obtained from various crops such as corn, rice, or potatoes or by chemically modifying the starch. Various types and degrees of chemical modification are used in the starch industry, and the labeling and use of chemically modified starches must meet government regulations.
Biosynthesis of starch is thought to occur through the action of four enzymes, ADP glucose pyrophosphorylase (EC 2.7.7.27), starch synthase (EC 2.4.1.21), starch branching enzyme (EC 2.4.1.18), and debranching enzyme (EC 2.4.1.41) [for reviews, see Smith, A. M., Denyer, K., and Martin, C. (1997) Annu. Rev. Plant Physiol. Plant Mol. Biol. 48:67-87; Myers, A. M. et al. (2000) Plant Phys. 122:989-997]. ADP glucose pyrophosphorylase catalyzes the synthesis of ADP glucose, the substrate for the synthesis of starch polymers. This enzyme is formed from a small and a large subunit. Most plants contain small multigene families of one or both of these subunits and in most cases various members of the family are differentially expressed in the plant organs. Starch synthase (SS), as its name implies, catalyzes the formation of α-1,4-linked glucose polymers from ADP-glucose. All plants possess granule bound starch synthases (GBSS) and most contain soluble starch synthases (SSI, SSII, SSIII). Branching enzymes (SBEs) catalyze the formation of amylopectin branch points by cleaving the α-1,4 linkages and creating new α-1,6 bonds. At least three SBE isoforms have been identified in maize. Debranching enzymes (DBEs) catalyze the hydrolysis of α-1,4 linkages and multiple isoforms have been found in all plants studied.
The proportion of amylose to amylopectin and the degree of branching of amylopectin are under genetic control. Differences in the degree of starch branching or polymerization are known to result in a change in the physiochemical properties of starch. Due to its unique functional properties, starch with high levels of amylose is in great demand in industry where high-amylose starches are high-value specialty products. These starches are very useful for industrial products because they form gels with high strength and have superior barrier and film-forming properties. In food uses, high-amylose starches readily form firm gels useful in confectionery products and contain resistant starch, which provides ingestible dietary fiber and is useful in low calorie food applications. Commercially available high-amylose starches from maize are extracted from grain containing the recessive ae mutation and are available in 2 classes. Class V is known as “50% amylose” and is sold under brand names such as Hylon® V and Gelose 50. Class VII is known as “70% amylose” and is sold under brand names such as Hylon® VII and Gelose 70.
In the last decade or so molecular genetic methods have been utilized to obtain plants producing starches with different ratios of amylose to amylopectin. In PCT publication No. WO 94/09144 (published 15 Jan. 1994) it is suggested that the use of sense and antisense transgenes may be used to alter the natural ratios of different starch synthase and branching enzymes in the recipient plant. This publication does not disclose any specific examples in which the starch characteristics were actually modified. PCT publication No. WO 00/06755 (published 10 Feb. 2000) shows the results of expressing all or a portion of a corn SBE isoform in sense or antisense orientation in transgenic corn. The resulting plants produce starches with higher levels of amylose and increased molecular weight of the amylose component together with shorter amylopectin chains than the non-transformed plants. Transgenic potato plants expressing, simultaneously, antisense versions of two potato SBE isoforms produce very-high-amylose starch (Schwall, G. P. et al. (2000) Nature Biotech. 18:551-554.
Alteration of starch fine structure in corn is complicated by the fact that three isoforms exhibiting starch branching enzyme activity have been identified in corn endosperm: SBEI, SBEIIa and SBEIIb. In the amylose extender (ae) mutant, SBEIIb activity is found to be deficient while in the dull (du) mutant, decreased levels of SBEIIa are observed (Boyer, C. D. and Preiss, J. (1981) Plant Physiol. 67:1141-1145). Later work has shown that the primary lesion in the dull mutation is a soluble starch synthase and deficiencies in this enzyme are responsible for the reduction in SBE (Gao, M. et al. (1998) Plant Cell 10:399-412). Studies of the catalytic properties of the corn starch branching enzymes indicate that the isoforms differ in substrate preference and in the length of glucan chain that is transferred. SBEI activity is higher when amylose serves as the substrate, and longer chains are preferentially transferred. The SBEII isoforms display higher activity with more highly branched substrates such as amylopectin. These enzymes preferentially transfer shorter glucan chains (Guan, H. and Preiss, J. (1993) Plant Physiol. 102:1269-1273; Takeda, Y. et al. (1993) Carbohydrate Res. 240:253-263). There is further evidence that the corn SBE isoforms are distinguished not only by their catalytic properties, but also by their pattern of expression in different corn tissues and in corn endosperm during development (Gao, M. et al. (1996) Plant Mol. Biol. 30:1223-1232; Gao, M. et al. (1997) Plant Physiol. 114:69-78).
By applying techniques of molecular biology, it has been possible to gain a better understanding of the role of individual SBE isoforms in starch biosynthesis and to generate unique starch phenotypes. An SBEIIa mutant obtained by Mutator insertional inactivation has recently been described. Endosperm starch isolated from this mutant lacks detectable SBEIIa and shows no change in amylopectin branch chain distribution. The amylose amounts were not reported in this study and no mention is made of the effect of combining the SBEIIa mutation with loss of either SBEI or SBEIIb (Blauth, S. L. et al. (2001) Plant Physiol. 125:1396-1405). Inhibition of SBEI expression alone produces no significant change in amylopectin structure or amylose content in corn starch. However, antisense inhibition of SBEI can be combined with the deficiency of SBEIIb in the ae mutant to generate an actual amylose level of about 50%, PCT publication WO 97/22703. This is compared to the actual amylose level of about 24% found in dent starch. The term “actual amylose” is defined below.
The measurement of amylose has long been a technical issue in the literature, and different measurement methods give remarkably differing results. The 50% amylose content of Class V starches and the 70% amylose content of Class VII starches is measured by iodine binding methods, which tend to overestimate the amount of amylose. For example, the double mutant amylose extender-waxy (aewx) starch from maize shows an amylose content of 15-26% when measured using the iodine binding method (Whistler, BeMiler, & Paschall in Starch: Chemistry and Technology 1984, p. 54). The overestimation in this method is made obvious because, in fact, this starch contains no amylose at all; all waxy starches lack all amylose as the waxy mutation results in a complete absence of functional granule bound starch synthase, the enzyme long known to be responsible for amylose biosynthesis.
An exception to the problem of overestimation of amylose levels using the iodine method are those of dent (wild type) starch and other normal or low amylose starches. The reason for this is simply that dent starch is used as a standard in these assays. The standards used for the calibration curve in these assays is prepared from dent and/or low amylose starches, which have an amylose content of about 23% to about 25%. With increasing amounts of amylose above that found in dent, the overestimation of amylose increases. This is due to mathematical reasons based on the use of dent starch as a standard and changes in amylopectin structure as an effect of branching enzyme inhibition, longer amylopectin chains are also formed and bind iodine, increasing apparent amylose.
The limitations of the old technique of amylose determination by iodine binding has long been recognized, and several other analytical methods have been proposed as superior. Fractionation of dispersed starch using differential precipitation by such solvents as thymol, n-butanol and/or isoamyl alcohol has been used both to prepare purified amylose and amylopectin, and to determine the amylose content of the starch. As is typical of most precipitation methods, absolute separations are very difficult to achieve. This technique also neglects the existence of ‘intermediate material’ which is present in significant amounts, especially in high amylose starches. (Klucinec, J. D. and Thompson, D. B. (1997) AACC Annual Meeting 223.163). This material is branched, and behaves in a similar manner to amylopectin in alcohol precipitation experiments, however it has been erroneously added to the amylose fraction in the past, again overestimating the amylose content. Other proposed techniques include Con A lectin precipitation, DSC determination of amylose-lipid binding, and size exclusion chromatography (SEC), using either native starch or starch debranched with isoamylase. With the sole exception of SEC of debranched starch these methods all give rise to overestimates of amylose content (Gerard, C. et al. (2001) Carbohydr. Polym. 44:19-27). While native starch GPC could theoretically be accurate, the difficulty in keeping the amylopectin in solution and its large radius of gyration makes this assay fraught with problems. Most practitioners of this method can recover only a small, non-representative proportion of the starting material from the column, particularly when sample preparation steps such as filtration (with pore size less than 5μ) or centrifugation are used. If the temperature and DMSO content of the mobile phase is not kept high throughout the sample preparation and chomatography steps, differential precipitation will distort the results.
A more practical, and accurate, method to measure the amylose content of starch is gel permeation chromatography (GPC) following enzymatic debranching of the amylopectin in the gelatinized starch. This allows complete loading of all carbohydrates from the starch, and quantitative recovery from the chromatography system. Values of amylose obtained in this fashion are referred to herein as “actual amylose.” Using this system, Class V starches (“50% amylose” by iodine methods) contain about 34% actual amylose and Class VII starches (“70% amylose” by iodine methods) contain about 42% actual amylose (whereas wild-type dent starches contain about 23-25% amylose). The starch of the present invention has an actual amylose level of at least about 76%.